Ubiquitin is an 8.5 kDa protein found in eukaryotic cells in either the free, monomeric state or covalently joined to various cytoplasmic, nuclear or membrane proteins. This protein contains 76 amino acid residues and its amino acid sequence is conserved to an unusually extent. The sequence of ubiquitin is identical between species as diverse as human, cow, Mediterranean fruit fly, Xenopus and chicken (U. Bond and M. Schlesinger (1985) Mol. Cell. Biol. 5: 949-956). Yeast and human ubiquitin differ by only three different amino acids (K. Ozkaynak et al. (1984) Nature 312: 663-666), while plant ubiquitin differs from that of yeast by two amino acids. Based on this two or three amino acid difference in sequence, there appear to be at least 3 types of ubiquitin--animal, plant and yeast.
Ubiquitin is found in three major cellular compartments--the cytoplasmic membrane, the cytoplasm and the nucleus. This protein is required for ATP--dependent degradation of intracellular proteins, a non-lysosomal pathway to eliminate from the cell those proteins that are damaged or abnormal as well as normal proteins having a short half-life (A. Hershko et al. (1984) Proc. Natl. Acad. Sci. USA 81: 1619-1623; D. Finley et al. (1985) Trends Biol. Sci. 10: 343-347). Ubiquitin binds to a target protein, tagging it for degradation. The covalent attachment is through isopeptide linkages between the carboxyl-terminus (glycine) in ubiquitin and the .epsilon.-amino group of lysyl side chains in the target proteins.
Ubiquitin also plays a role in the cellular response to stresses, such as heat shock and increase in metal (arsenite) concentration (D. Finley et al. (1985) supra). Most living cells respond to stress (for example, exposure to temperatures a few degrees above normal physiological temperatures or to elevated concentrations of heavy metals, ethanol, oxidants and amino acid analogs) by activating a small set of genes to selectively synthesize stress proteins, also called heat shock proteins. In most organisms these stress proteins were found to have subunit molecular weights of 89, 70 and 24 kDa (U. Bond and M. Schlesinger (1985) supra). Ubiquitin, with a molecular weight of approximately 8.5 kDa, also responds to stress, since in different species (yeast, mouse, gerbil and chicken embryo fibroblasts) the levels of ubiquitin mRNA and ubiquitin protein increase as a result of different stress conditions.
In eukaryotic systems the expression of genes is directed by a region of the DNA sequence called the promoter. In general, the promoter is considered to be that portion of the DNA, upstream from the coding region, that contains the binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region also comprises other elements that act as regulators of gene expression. These include a TATA box consensus sequence in the vicinity of about -30, and often a CAAT box consensus sequence at about -75 bp 5' relative to the transcription start site, or cap site, which is defined as +1 (R. Breathnach and P. Chambon (1981) Ann. Rev. Biochem. 50: 349-383; J. Messing et al. (1983) in Genetic Engineering of Plants, eds. T. Kosuge, C. P. Meredith and A. Hollaender, pp. 211-227). In plants the CAAT box may be substituted by the AGGA box (J. Messing et al. (1983) supra). Other regulatory elements that may be present are those that affect gene expression in response to environmental stimuli, such as illumination or nutrient availability, or to adverse conditions, such as heat shock, anaerobiosis or the presence of heavy metal. In addition, there may be present DNA sequences which control gene expression during development, or in a tissue-specific fashion. Other regulatory elements that have been found are the enhancers (in animal systems) or the upstream activating sequences (in yeast), that act to elevate the overall expression of nearby genes in a manner that is independent of position and orientation with respect to the nearby gene. Sequences homologous to the animal enhancer core consensus sequence, 5'-GGTGTGGAAA(orTTT)G-3', have been described in plants, for example, in the pea legumin gene at about position -180 relative to the transcription start site (G. Lycett et al. (1984) Nucleic Acids Res. 12: 4493-4506) and in the maize Adh1 and Adh2 genes at about -200 and -170 bp, respectively, from the transcription start site. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding gene and this promoter region, comprising all the ancillary regulatory elements, may contain between 100 and 1000 or more nucleotides.
Of the regulatory elements controlling gene expression, the heat shock element is perhaps one of the most widely studied. Although the universality of cellular response to heat shock has been known for almost a decade, very little is known yet about the function of the heat shock proteins selectively synthesized by the stressed cell. The induction of stress protein synthesis occurs at a transcriptional level and the response has been found to be similar in bacteria, fungi, insects and mammals (E. Craig (1985) CRC Crit. Rev. Biochem. 18: 239-280). In addition to the synthesis and accumulation of the classic heat shock proteins in response to stress, cells that are stressed also synthesize proteases and ubiquitin. In E. coli, a 94 kDa enzyme that has ah ATP-dependent proteolytic activity is encoded by the lon (cap R) gene whose expression is under control of the heat shock regulon (E. Ozkaynak et al. (1984) Nature 312: 663-666). In chicken embryo fibroblasts (U. Bond et al. (1985) Mol. Cell. Biol. 5: 949-956) the ubiquitin mRNA level increased five fold after heat shock or after exposure to 50 .mu.M arsenite. Each mRNA comprises tandemly repeated identical polypeptides which, upon translation as a polyubiquitin molecule, gives rise to multiple ubiquitin molecules, offering a distinctive mechanism for amplifying genetic information. This elevated level of ubiquitin mRNA does not persist during the recovery phase after heat shock, indicating a transient role for free ubiquitin during the stress response.
It has been postulated (J. Ananthan et al. (1986) Science 232: 522-524) that metabolic stresses that trigger the activation of heat shock protein genes act through a common mechanism. The metabolic stresses that activate heat shock genes cause denaturation of intracellular proteins; the accumulation of abnormal proteins acts as a signal to activate heat shock genes. A role for ubiquitin in targeting abnormal proteins for degradation, as well as for different proteolytic enzymes, would be compatible with such a model of heat shock protein gene regulation.
Most of the early work on heat shock genes was done with Drosophila species. In particular, the Drosophila hsp70 gene was used widely in recombinant studies. In homologous systems, the Drosophila hsp70 gene was fused to the E. coli .beta.-galactosidase structural gene to allow the activity of the hybrid gene to be distinguished from the five resident hsp70 heat shock genes in the recipient Drosophila. Drosophila heat shock genes were also introduced into heterologous systems, e.g., in monkey COS cells and mouse cells (H. Pelham (1.982) Cell 30: 517-528). Regulation by heat shock was observed in the hybrid hsp70lac Z gene which was integrated into the Drosophila germ line and into which a 7 kb E. coli .beta.-galactosidase DNA fragment was inserted into the middle of the hsp70 structural gene. The resultant .beta.-galactosidase activity in the transformants was shown (J. Lis et al. (1983) Cell 35: 403-410) to be regulated by heat shock.
The DNA sequence conferring heat shock response was identified by deletion analysis of the Drosophila hsp70 heat shock promoter to be 5'-CTGGAAT.sub.-- TTCTAGA-3' (H. Pelham et al. (1982) in Heat Shock From Bacteria to Man, Cold Spring Harbor Laboratory, pp. 43-48) and is generally located in the -66 through -47 region of the gene or approximately 26 bases upstream of the TATA box. It was further demonstrated that a chemically synthesized copy of this element, when placed upstream of the TATA box of the herpes virus thymidine kinase gene in place of the normal upstream promoter element, was sufficient to confer heat inducibility upon the thymidine kinase gene in monkey COS cells and in Xenopus oocytes. (The thymidine kinase gene is normally not heat inducible.) These heat shock sequences interact with heat shock specific transcription factor(s) which allow the induction of heat shock proteins (C. Parker et al. (1984) Cell 37: 273-283). Inducers of heat shock genes could be factors that alter (decrease) the concentration of heat shock proteins within the cell and, thus, control the transcription and translation of heat shock genes.
In higher plants, the stress response was demonstrated by increased protein synthesis in response to heat shock in soybean, pea, millet, corn, sunflower, cotton and wheat (T. Barnett et al. (1980) Dev. Genet. 1: 331-340; J. Key et al. (1981) Proc. Nat. Acad. Sci. USA 78: 3526-3530). The major differences in heat shock response seen among plant species are: (a) the amount of total protein synthesized in response to stress, (b) the size distribution of the different proteins synthesized, (c) the optimum temperature of induction of heat shock proteins and (d) the lethal (breakpoint) temperature. High molecular weight proteins are found to be electrophoretically similar among different species. The low molecular weight (15-27 kDa) heat shock proteins show more electrophoretic heterogeneity between species. In plants, the higher molecular weight proteins resemble those produced in Drosophila. There is a marked difference, however, in the complexity of the low molecular weight heat shock proteins between plants and Drosophila. Four heat shock proteins, 22, 23, 26 and 27 kDa, are synthesized in Drosophila, whereas soybean produces over 20 heat shock proteins having molecular weights in the range of 15-18 kDa. The low molecular weight protein genes in soybeans are the most actively expressed and coordinately regulated genes under heat shock conditions (F. Schoffl et al. (1982) J. Mol. Appl. Genet. 1: 301-314).
Key et al. (U.S. patent application Ser. No. 599,993, filed Apr. 13, 1984) have studied the promoter region of plant heat shock genes. Four soybean heat shock genes (three genes coding for 15-18 kDa heat shock proteins and one gene coding for a 173 kDa heat shock protein) were cloned and sequenced. The coding sequences and flanking sequences of the four heat shock genes were determined. The promoter regions of these four genes were subcloned, linked to a T-DNA shuttle vector and transferred into Agrobacterium tumefaciens. One of the recombinant clones of a soybean heat shock gene coding for a 15-18 kDa protein contained an open reading frame of 462 nucleotides and a 291 nucleotide promoter region upstream of the ATG translation initiation codon. The promoter included the TATA box, the CAAT box, the transcription initiation site and a heat shock consensus sequence 131-144 nucleotides upstream of the ATG translation start codon with the sequence 5'-CT.sub.13 GAA.sub.13 TTC.sub.13 AG-3'. Only three of the four clones showed substantial homology in the promoter region, but there were strong similarities between the heat shock consensus sequences of all four clones. Significantly, the coding sequence, the upstream promoter region and the downstream flanking region of the four soybean heat shock genes had almost no resemblance to the corresponding regions of Drosophila heat shock genes. Although there were similarities between the consensus sequence of the promoter region from Drosophila and soybean heat shock genes, the promoter regions of soybean heat shock genes did not possess the inverted repeat sequences characteristic of Drosophila genes.
The promoter region from the soybean heat shock genes was used to activate a soybean gene and a foreign gene (one normally not found in soybean) and to show regulation of the response by stress (Key et al. U.S. patent application Ser. No. 599,993, filed Apr. 13, 1984). The promoter was isolated from the soybean SB 13 heat shock gene as a DNA fragment extending 65 bp downstream from the start of transcription to include a major portion of the untranslated leader sequence but not the start codon for translation. A .beta.-galactosidase gene was placed under the control of the heat shock promoter within the T-DNA of the Ti-plasmid in a stable form within A. tumefaciens and then was transferred to a plant or plant cell culture. The actuality of DNA transfer was recognized by the expression of the .beta.-galactosidase gene as the production of a blue color after heat treatment in a medium containing the 5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside substrate molecule (M. Rose et al. (1981) Proc. Natl. Acad. Sci. USA 78: 2460-2464).
Experimentation with cross expression wherein a gene from one plant species is examined for expression in a different species adds a further dimension to the understanding of specific function. These experiments may embody the insertion of a gene under the control of its own promoter or of a gene artificially fused to a different or unnatural promoter. In 1983 Murai et al. (Science 222: 476-482) obtained expression of the phaseolin gene from Phaseolus vulgaris L. in sunflower (Helianthus) tissue under two sets of conditions: (i) when the Phaseolin gene was under the control of its own promoter and (ii) when the gene was spliced to, and under the control of a T-DNA promoter. In subsequent experiments it was shown that the phaseolin structural gene under the control of its natural promoter could be expressed in tobacco and that the tissue-specific expression in the heterologous host (tobacco) was similar to that in the native host (bean) (C. Sengupta-Gopalen et al. (1985) Proc. Natl. Acad. Sci. USA 82: 3320-3324).
In later experiments (J. Jones et al. (1985) EMBO J. 4: 2411-2418) the expression of the octopine synthetase gene (ocs) was described in both regenerated transformed homologous (petunia) and heterologous (tobacco) plants. In this study the ocs gene was fused to the promoter of a petunia chlorophyll a/b binding protein. Cross-expression was also obtained by W. Gurley et al. (1986) (Mol. Cell. Biol. 6: 559-565) and Key et al. (U.S. patent application Ser. No. 599,993, filed Apr. 13, 1984), who reported strong transcription in sunflower tumor tissue of a soybean heat shock gene under control of its own promoter. In this case functional activity was measured as the correct thermal induction response.
The first evidence for transcription initiated from a monocotyledon promoter in a dicotyledon host plant was published by Matzke et al. (1984) (EMBO J. 3: 1525-1531). These workers cloned the maize zein Z4 gene and introduced it on a Ti-derived vector into sunflower stemlets. The ensuing zein mRNA could then be translated in a wheat germ system but not in the transformed sunflower calli.
In a later study the wheat gene whAB1.6 encoding the major chlorophyll a/b binding protein was cloned into a T-DNA-containing vector and transferred to both petunia and tobacco (G. Lamppa et al. (1985) Nature 316: 750-752). Expression was obtained in both the monocotyledon and dicotyledon hosts and was determined to be light-induced and tissue-specific. In a more recent study, Rochester et al. (1986) EMBO J. 5: 451-458) obtained expression of the maize heat shock hsp70 gene in transgenic petunia. The maize hsp70 mRNA was synthesized only in response to thermal stress. So far, these three studies constitute the total number of published reports describing-successful expression of monocot genes in transgenic dicot plants. However, there are also negative reports describing minimal or no expression of maize alcohol dehydrogenase gene in tobacco hosts (Llewellyn et al. (1985) in Molecular Form and Function of the Plant Genome, L. van Vleten-Doting, G. S. Greet and T. Hall (eds), Plenum Publishing Corp., pp 593-608; J. G. Ellis et al. (1987) Embo J. 6: 11-16) , suggesting a possible inherent species-specific difference between monocot and dicot promoters.
The heat shock response is believed to provide thermal protection or thermotolerance to otherwise nonpermissive temperatures (M. Schlesinger et al. (1982) in Heat Shock from Bacteria to Man, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 329). A permissive heat shock temperature is a temperature which is high enough to induce the heat shock response but not high enough to be lethal. Thermotolerance in plant seedlings can be attained by different treatment regimes: (a) a 1 to 2 hour exposure to continuous heat shock at 40.degree. C. followed by a 45.degree. C. incubation, (b) a 30 min heat shock at 40.degree. C. followed by 2 to 3 hours at 28.degree. C. prior to the shift to 45.degree. C., (c) a 10 min heat shock at 45.degree. C. followed by about 2 hours at 28.degree. C. prior to the shift to 45.degree. C. and (d) treatment of seedlings with 50 .mu.M arsenite at 28.degree. C. for 3 hours or more prior to the shift to 45.degree. C. During the pretreatment prior to incubation at the potentially lethal temperature, heat shock proteins are synthesized and accumulated. Also, heat shock mRNA and protein syntheses occur at 45.degree. C., if the plant seedling is preconditioned as described above. When the temperature is shifted back to physiological levels (e.g., 28.degree. C.), normal transcription and translation are resumed and after 3 to 4 hours at normal temperature, there is no longer detectable synthesis of heat shock proteins (J. Key et al. (1981) Proc. Natl. Acad. Sci. USA 78: 3526-3530; M. Schlesinger et al. (1982) Trends Biochem. Sci. 1: 222-225). The heat shock proteins that were synthesized during the 40.degree. C. heat shock treatment are very stable and are not immediately degraded.
Although ubiquitin is regulated in response to environmental stress, including heat shock, the regulation of ubiquitin transcription differs from that of classical heat shock protein transcripts. Both ubiquitin and heat shock protein mRNA levels are elevated in response to cellular stress. However, whereas classical heat shock proteins accumulate during heat shock and persist during the recovery phase, ubiquitin mRNAs accumulated during heat shock are rapidly degraded within hours after stress treatment. This unstable mRNA transcript suggests a specialized but transient role for ubiquitin during heat shock, and implicates a unique DNA sequence in the ubiquitin gene promoter region, specifying specialized regulatory control during cellular response to stress.